It has long been known that it may be advantageous to produce desired proteins as fusions, especially with amino acid sequences homologous to the expression host. For example, LaVallie, E. R., et al., Current Opinion in Biotech (1995) 6:501-506 provides a summary and review of the use of expression of fusions of desired proteins in E. coli to overcome the problems associated with bacterial expression, such as the formation of inclusion bodies (which require refolding schemes) or failure of E. coli completely to remove the amino terminal methionine initiation codon. Fusion proteins have also been employed in plants with mixed results. For example, Parmenter, D. L., et al., Plant Mol. Biol. (1995) 29:1167-1180 describe the production of the anticoagulant protein, hirudin, as a fusion with the seed-specific protein, oleosin. Oleosins are small proteins and are imbedded in the phospholipid monolayer of oil bodies, and thus the fusions are relatively easy to purify. Successful production of hirudin in rape seed was achieved using this strategy. Successful fusion of the oleosin coding sequence with the heterologous β-glucuronidase in Brassica napus seeds was also demonstrated by Van Rooijen, G. J. H., et al., Bio/technology (1995) 13:72-77. On the other hand, while Vendekerckhove, J., et al., Bio/technology (1989) 7:929-932 produced the foreign protein Leu-enkephalin in B. napus and Arabidopsis seeds using a fusion with the 2S seed storage albumin protein, it appears that this was less successful due to the impact of the fusion on the post-translational fate of the modified seed storage protein.
Particularly relevant is the report by Sardana, R. K., et al., Transgenic Res. (2002) 11:521-531 which reports the production of human granulocyte-macrophage colony stimulating factor (GM-CSF) in transgenic tobacco plants under the control of the rice endosperm-specific glutelin promoter Gt3 to produce a fusion with the eight N-terminal amino acids of glutelin. It appeared that protein extracts from plants with either the construct comprising the eight glutelin amino acids or GM-CSF alone were biologically active.
While the present invention relates to fusions for production of desired proteins in general in plants, a particular application is the use of such fusion proteins to improve nutritional content. Plant proteins are relatively low in the nine essential amino acids required by mammalian consumers. Thus, for example, the nutritional value of cereal grain proteins is a critical constraint in their uses for both human food and animal feed, as they are mostly deficient in lysine and other essential amino acids (Sun, S. S. M., et al., Transgenic Plants (1993) 1:339-371). Rice, a low cost energy and protein source and the staple food of over half of the world's population, is relatively unbalanced in essential amino acid content, with lysine being the first limiting essential amino acid. It is costly and sometimes not feasible to supplement the cereal seeds with crystalline lysine or other nutritionally balanced proteins to correct for the deficiency. Therefore, improvement of rice seed proteins for increased lysine content is of extreme importance for those who rely upon rice as main staple food.
Significant effort has been made in the past to improve the quality (lysine content) of cereal grain proteins, e.g., maize opaque-2 (o2) mutant (Mertz, E. T., et al., Science (1964) 145:279-280). Unfortunately, undesirable traits often associated with such modified crops, such as lower yields and greater susceptibility to pests and diseases, preventing their agronomic utilization. Similarly, efforts in breeding rice containing increased lysine have not met with significant success. Recently with progress in biotechnology, enhancement of the nutritional value of crops has been shown to be feasible by introducing heterologous or modified genes encoding storage proteins rich in essential amino acids. For example, the contents of sulfur-containing amino acids, methionine and cysteine, but not of lysine, have been enhanced in legume and other crops, especially in cereal crops (Sun, S. S. M., et al., In Vitro Cell. Dev. Biol.-Plant (2004) 40:155-162).
Cloning of cDNA encoding a 18-kDa lysine-rich protein (LRP) from winged bean (Psophocarpus tetragonolobus) is disclosed in Sun, S. S. M., et al., U.S. Pat. No. 6,184,437. With a 10.7 mol % lysine content, this protein has great potential for improving the content of lysine in cereal crops. More recently, LRP has demonstrated to be tissue-specifically expressed in the seeds of transgenic rice plants under the control of the rice glutelin Gt1 promoter, and stably accumulated in the mature seeds of transgenic rice (Liu, et al., unpublished). The accumulation level of LRP in transgenic rice seeds was not particularly high, however, amounting only to about 1% of the total seed storage protein even in the highest expression lines, resulting in only a few % increase in lysine content in the dry rice seeds. Therefore, further lysine enhancement is required for effective improvement of the nutritional value of cereal grains.
Even if use of heterologous proteins could be achieved by enhancement at transcriptional level, as by strong seed-specific promoters, barriers still exist with regard to the various posttranscriptional steps required to produce a stable mature protein, such as, for example, conformation and subcellular targeting and deposition. In previous studies, we had attempted to enhance the expression of LRP gene by using a stronger promoter and/or fusing with signal peptide sequences of rice storage protein genes, but did not observe significant increase in the yield of protein in the mature transgenic rice seeds (Liu, et al., unpublished).